Mycobacteria Flashcards by Antonio Flamenco

important human pathogens of Mycobacteria

○ Mycobacterium tuberculosis complex (MTBC)
○ Nontuberculosis mycobacteria (NTM)
○ Mycobacterium avium-intracellulare complex (MAI)

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What are mycobacteria considered?

○ BSL 3
○ recommended .to reduce the risk of inhalation through aerosolization

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Describe Mycobacterium

○ Slender, slow growing bacilli
○ Obligate aerobes
○ Cell division occurs by branching

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How do you enhance growth of Mycobacterium?

Growth enhanced with CO2 (10%)

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What is in the cell wall of Mycobacterium?

○ ~60% of the cell wall is comprised of lipids and lipid containing structures
○ Mycolic acid and Cord factor

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Mycolic acid

○ strong hydrophobic molecules that form a lipid shell around the organism
○ affect permeability properties at the cell surface

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What is Mycolic acid an important virulence factor for?

Mycobacterium tuberculosis

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Cord Factor

○ responsible for the serpentine cording
○ toxic to mammalian cells
○ an inhibitor of PMN migration

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Where is cord factor main made?

virulent strains of Mycobacterium tuberculosis

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What do the high concentration of lips contribute to Mycobacterium tuberculosis?

○ Resistance to many antibiotics
○ Resistance to killing by acidic and alkaline compounds
○ Resistance to osmotic lysis
○ Resistance to lethal oxidations and survival inside of macrophages
○ Impermeability to stains and dyes

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Acid-fast bacilli (AFB)

Mycobacteria that are difficult to decolorize when using as acid alcohol

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What are the 3 staining methods for mycobacteria?

○ Ziehl-Neelsen technique
○ Kinyoun Technique
○ Fluorochrome Stain

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Ziehl-Neelsen technique

○ smear is fixed and stained with hot carbol-fuchsin
○ decolorized with acid-alcohol
○ counterstained with methylene-blue
○ Acid fast bacilli appear pink in a contrasting background
○ Also this is no longer used

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Kinyoun Technique

○ Similar to Ziehl-Neelsen technique but uses detergents to drive the dye into the cell wall instead of heat
○ Similar in appearance (pink contrast to background)

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Fluorochrome Stain

○ uses auramine O as a primary stain
○ rhodamine as the counter stain

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Fluorochrome stain fact

○ stains are more sensitive
○ more labor intensive and must be read using a fluorescent microscope

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mycobacteria collection?

○ Sputums
○ Bronchial washes
○ Transtracheal aspirates

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When do you collect mycobacteria samples? How many?

○ 3 samples
○ early morning preferred
○ for 3 consecutive days

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What if a sample collection of sputum isn’t available? Like in children

Gastric lavage, early in the morning

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What is done to urine samples of mycobacteria?

Centrifuged to concentrate

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What fluids are collected for mycobacteria?

○ CSF
○ synovial fluid
○ pericardial fluid
○ peritoneal fluid

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For blood samples, what is used to release the bacteria from red/white blood cells?

Lysing agents (saponin)

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What is needed for mycobacteria processing of respiratory samples?

Decontamination and digestion

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What is used for decontamination? What does it do?

○ NaOH or benzalkonium chloride
○ Kills normal respiratory flora or pathogens
○ reduces contaminants

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How does NaOH work?

increases pH to a level that is antibacterial

What is used for digestion? What does it do?

○ N-acetyl-L-cysteine (NALC) ○ Liquefies mucus in a respiratory sample which may trap mycobacteria

What is needed to grow mycobacteria?

whole egg

What is the traditional growth media for mycobacteria?

Lowenstein-Jensen Media

Describe Lowenstein-Jensen Media

○ Prepared as agar slants ○ Malachite green (inhibitor) ○ Incubation 6-10 weeks ○ Can see growth as early as 18-24 days

What is the agar based media to grow mycobacteria?

Middlebrook 7H10 or Middlebrook 7H11

Describe Middlebrook 7H10/7H11

○ transparent ○ contains 2% glycerol to support the growth of MAC ○ Growth detected between 10-12 days

What is Middlebrook 7H9 broth?

○ nonselective liquid media ○ maintains stock cultures ○ prepares isolates for biochemical analysis

What is Mycobacterium growth index tube (MGIT)

○ modified 7H9 broth with a fluorchrome ○ Glows under UV light if positive

What are the incubation temperatures for mycobacteria species?

○ MTB 35°C – 37°C ○ M. marinum and M. ulcerans 30°C - 32°C ○ M. xenopi 42°C – 45°C

Are what CO2 level are mycobacterium kept at?

10%

What does Mycobacterium Tuberculosis cause?

As the name implies, tuberculosis

How is Mycobacterium Tuberculosis transmitted?

respiratory droplets containing the organism

Once it's inhaled, where does Mycobacterium Tuberculosis deposit?

bronchiole or alveoli

For immunocompromised patients, where does Mycobacterium Tuberculosis spread?

○ lymph nodes ○ Spine ○ long bone ○ Heart ○ Meninges ○ gastro-urinary system

Mantoux tuberculin skin test (TST)

○ used to determine if a person has been exposed to MTB organisms ○ Uses PPD that is injected into the forearm

or Mantoux tuberculin skin test (TST), what causes false positive reactions?

Calmette-Guerin (BGC) vaccination

For Mantoux tuberculin skin test (TST), what causes false negative reactions?

○ Suppressed immune system can cause skin test to not react ○ Recent or very old infection ○ Infants less than 6 months of age with an overwhelming TB disease

For TST what is recommended for healthcare workers?

○ Two step testing is recommended ○ second TST is administered if first is negative after 1-3 weeks ○ If both tests are negative, person is fine

QuantiFERON TB Gold In-Tube test (QFT-GIT)

Looks for antibodies produced from past or present infection of TB

T-SPOT TB test (T-Spot)

○ Blood tests to detect TB infection ○ Can detect latent TB (nonactive TB)

How does T-SPOT TB test (T-Spot) work?

When WBCs are infected with MTB a substance interferon – gamma (IFN-γ) is released and can be detected

For MTB, what are some identifiers on LJ or Middlebrook media?

○ tan to buff colonies in 14-28 days at 35-37°C ○ Colony morphology is rough, dry, warty and granular (looks like breadcrumbs or cauliflower)

What are some 1st line drugs against MTB?

○ Isoniazid (INH) ○ Rifampin ○ Pyrazinamide ○ Ethambuto

What are some 2nd line drugs against MTB?

○ Streptomycin ○ Cycloserine ○ P-aminosalicylic acid ○ Ethionamide

What is the mycobacterium tuberculosis complex?

Genetically related group of Mycobacterium species that can cause tuberculosis in humans or other organisms

What organisms are a part of the mycobacterium tuberculosis complex?

○ M. tuberculosis ○ M. africanum ○ M. canettii ○ M. bovis ○ M. microti ○ M. caprae ○ M. orygis ○ M. pinnipedii

Mycobacterium avium complex (MAC) consists of two species:

M. avium and M. intracellulare

Why are the two species of MAC also called Mycobacterium avium-intracellulare (MAI)?

Because these species are difficult to differentiate

Mycobacterium avium-intracellulare (MAI)

atypical Mycobacterium most commonly associated with human disease

MAC is primarily a what?

○ pulmonary pathogen ○ affects individuals who are immune compromised

How would you Identify MAC group?

○ acid-fast bacillus (AFB) ○ Colony morphology is smooth ○ tan to buff colonies in 14-28 days at 35-37°C

M. ulcerans

○ produces cutaneous lesions called Buruli ulcers ○ Appear as lumps under the skin ○ non-healing ulcers

M. ulcerans identification?

Flat, rough, pale colonies on LJ and Middlebrook agars at 32-33°C

M. bovis

Agent of tuberculosis in cattle and other warm-blooded animals

How has M. bovis been handled?

○ better pasteurization techniques ○ strict guidelines on cattle handling and management by FDA

Bacillus Calmette–Guérin (BCG) vaccine

vaccine primarily used against tuberculosis in countries where tuberculosis is common

M. leprae

Cause of Hansen’s disease or leprosy

How do you grow M. leprae? And why?

○ grown on the foot pads of mice ○ Cannot be cultured in vitro

M. Leprae Characterized by?

○ skin sores and nerve damage ○ Can get worse without treatment: lesions on the skin numbness in the extremities muscle weakness

M. leprae Identification?

AFB in characteristic skin lesion specimens